Pancreatic cancer is the fourth leading cause of cancer death in the US. It is also one of the deadliest tumors with a median survival of less than six months and few effective treatment options. Thus, even a modest improvement in the therapeutic response rate can be expected to have a significant impact on patient survival. Among the few chemotherapeutic drugs that show some efficacy is 5-fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor that has been in use for nearly 50 years. Studies have shown that high tumor levels of TS are a poor prognostic indicator, and are associated with resistance to 5-FU. Conversely, reducing cellular TS levels sensitizes tumor cells to 5-FU and other TS inhibitors, thereby improving the drugs'efficacy. A novel way to suppress TS activity has been identified based on our finding that an active-site double-mutant (DM) of TS functions as a dominant-negative inhibitor of TS when it interacts with the wild-type enzyme. When the human DM-TS is expressed in cancer cells, TS activity is reduced and the cells are more sensitive to 5-FU. Here, we propose to exploit these findings to enhance the drug sensitivity of pancreatic cancer by impairing this key enzyme. The hypothesis is that the selective attenuation of TS activity in tumor cells with a dominant-negative DM-TS will sensitize the cells to 5-FU and other TS inhibitors, thereby increasing the effectiveness of the chemotherapy. Short dominant-negative peptides derived from the DM-TS have been identified that effectively inhibit cellular TS activity, and ways to efficiently introduce these peptides into pancreatic tumor cells will be developed. This approach will be tested in pancreatic tumor cell lines, as well as in actual pancreatic tumor tissue explants in culture. It is expected that treating tumors with a dominant-negative peptide will increase the efficacy of TS inhibitor-based chemotherapy, both in single- and in combination-drug regimens. These experiments should demonstrate that dominant-negative inhibition of TS can be used to sensitize pancreatic cancer cells to chemotherapy, and lay the groundwork for the further development of this treatment modality for use in vivo in animal models and ultimately in humans. PUBLIC HEALTH RELEVANCE: According to the American Cancer Society, over 30,000 new patients will be diagnosed with pancreatic cancer in 2009, and almost as many will die from it. Indeed, pancreatic cancer is the 4th most cause of cancer related death, and few if any effective treatment options exist. Therefore, it is imperative for the scientific community to come up with new approaches for the treatment of this deadly disease. The research proposed by us has as its goal to directly improve the efficacy of chemotherapy for pancreatic cancer, and thus is in line with the goal of the Healthy People 2010 initiative to "Reduce the number of new cancer cases as well as the illness, disability, and death caused by cancer", and the mission of the National Cancer Institute to "eliminate suffering and death due to cancer"'by the year 2015.